This R21/R33 Phased Innovation Award application proposes to develop novel RANTES derivatives capable of recombinant expression as candidate topical microbicides. These molecules target the CCR5 coreceptor used by HIV-1 isolates responsible for primary transmission. Two lead candidates with antiviral activity against CCR5-using HIV-1 isolates equivalent to the fully synthetic PSC-RANTES have been identified using an innovative phage display selection process that allows screening of millions of CCR5 binding molecules. One of these molecules shares the inhibitory mechanism of PSC-RANTES, CCR5 internalization and sequestration, and also shares signaling activity via CCR5. The second molecule, although equally potent at inhibiting virus infection, does not cause CCR5 internalization or signaling via CCR5. These properties could improve the safety profile of this molecule. The objectives of the R21 phase of the proposal are to determine the mechanism of activity of the two molecules, the duration of activity after compound removal, and the activity against clade A, C, and D primary transmission isolates from sub- Saharan Africa. We will also attempt to generate resistant variants to both molecules in vitro, and compare the stability of the molecules to low pH, high temperature, and exposure to cervicovaginal lavage (CVL) fluid. The molecule with the best combination of properties in these experiments will be selected for preclinical evaluation in the R33 phase using the SHIV162P3 vaginal challenge model in female rhesus macaques. These experiments will establish the effective dose for preventing transmission, determine if that dose protects against delayed virus challenge, and evaluate local inflammation and induction of proinflammatory cytokines as indicators of safety. Virus recovered from treated animals with delayed viremia will be evaluated for development of resistance to the lead RANTES derivative. These studies are designed to lead to a safe, effective, and inexpensive protein CCR5 inhibitor for incorporation into a topical microbicide for use in areas most impacted by the global HIV/AIDS pandemic. [unreadable] [unreadable] [unreadable]